Euglena: 2013 Xanthophyta (Allorge ex Fritsch 1935) and Bacillariophyta (Haeckel 1878) are basal groups within Ochrophyta (Cavalier-Smith 1986) Hannah Airgood, Jason Long, Kody Hummel, Alyssa Cantalini, DeLeila Schriner Department of Biology, Susquehanna University, Selinsgrove, PA 17870. Abstract Xanthophyta and Bacillariophyta are phyla within Heterokontae. Our focus was to examine the topology of Ochrophyta, the photosynthetic heterokonts, especially the position of Xanthophyta and Bacillariophyta. Two similar 28S rRNA genes and a third 18S rRNA gene were used for molecular analysis, by maximum likelihood. Fucoxanthin in chloroplasts, symmetry, and number of flagella were the characters used for morphological analysis. We concluded that Xanthophyta is a basal group in Ochrophyta. Bacillariophyta were also shown to be basal within Ochrophyta after Xanthophyta. In our study we found a misidentified species. A third gene was used to confirm this misidentification. Please cite this article as: Airgood, H., J. Long, K. Hummel, A. Cantalini, and D. Schriner. 2013. Xanthophyta (Allorge ex Fritsch 1935) and Bacillariophyta (Haeckel 1878) are basal broups within Ochrophyta (Cavalier-Smith 1986). Euglena. doi:/euglena. 1(2): 43-51. Introduction phaeophytes as they state that they are sister groups Heterokontae (Cavalier-Smith 1986) based on fossil analysis of xanthophytes and includes the phylum Ochrophyta, which is a phaeophytes. Riisberg et al. (2009) show evidence of monophyletic group of photosynthetic taxa. The the diatoms being basal to all the groups analyzed united group is further divided into Xanthophyta and that xanthophyte was more recently derived. It is (Allorge ex Fritsch 1935), Phaeophyta (Kjellman important that there are some diatoms that have radial 1891), Raphidiophyta (Chadefaud 1950), symmetry, but the evolution of bilateral symmetry in Chrysophyta (Pascher 1914), Eustigmatophyta this group is significant (Holt and Iudica 2012). (Hibberd and Leedale 1970), Silicoflagellata (Borgert One of the characters that unite heterokonts 1890), Pinguiophyta (Kawachi 2002), and is the presence of two heterodynamic flagella Bacillariophyta, the diatoms (Haeckel 1878). Three (Phillips et al. 2008), the anterior one of which has characters that are significant to understanding the tripartite tubular hairs. Pinguiophyta are separate topology of ochrophytes are the presence or absence from the other taxa with a single flagellum (Andersen of fucoxanthin, symmetry, and number of flagella 2004), having lost the second flagellum. The (Andersen 2004, Negrisolo et al. 2004 and Holt and presence of two basal bodies found within Iudica 2012). Fucoxanthin in the chloroplasts is an pinguiophytes indicates the past presence of a second important character as the absence of this character flagellum (Andersen 2004). distinguishes and separates xanthophytes from the The purpose of the paper was to show the other taxa (Andersen 2004 and Negrisolo et al. 2004). position of xanthophyte as a basal group to all Historically, xanthophytes has been difficult to place. photosynthetic heterokonts. They have been classified as their own phylum by Margulis and Schwartz (1998). They have also been Materials and Methods placed within chrysophyte (Lee 1999). Beakes (1989) A collection of 31 in-group species was demonstrated that xanthophytes are related to selected to analyze the evolutionary relationship oomycetes. The placement of xanthophyte is still among the Ochrophyta. The taxa include 5 species of unclear, with new work done by Yang et al. (2012) Chrysophyta, 2 species of Pinguiophyta, 1 species of suggesting that xanthophytes are sisters to Eustigmatophyta, 5 species of Silicoflagellata, 3 phaeophytes. species of Raphidiophyta, 6 species of Phaeophyta, 5 Cavalier-Smith et al. (2005) studied the 28S species of Bacillariophyta, and 4 species of rDNA gene and indicated a close relationship Xanthophyta (Appendix A). Caecitellus parvulus between phaeophytes, xanthophytes and (Paterson, Nygaard, Steinberg, Turley 1993), a raphidiophytes. Brown and Sorhannus (2012) support Bicosoecida (Grasse 1926), was used as an out- the relationship between xanthophytes and group. Two similar genes were used for the 43 Euglena: 2013 molecular analysis of these species (Accession with Invariant sites (G+I), seen in Figure 4. The numbers: FJ030880 + FJ030881). These genes were values seen on the trees are the bootstrap values for 28S rRNA genes from Riisberg et al. (2009). The which are obtained by making 1000 iterations of the sequences obtained from the NCBI database were tree and analyzing the clades in each tree. The values placed in MEGA 5.1 (Tamura et al. 2011) where they are the frequency that the particular clade is seen in were aligned, using ClustalW, and trimmed to obtain those 1000 iterations. sequences of 9029 bases. The aligned sequences were These trees were combined to form a then analyzed using 4 maximum likelihood trees, consensus tree, Figure 5. The character evolution, each with different models, using 1000 bootstrap seen in Table 1, was also mapped on this tree. The replications (Figures 1-4). characters were fucoxanthin in the chloroplasts, A model analysis was first performed to symmetry, and number of flagella. obtain different models for maximum likelihood Further analysis was performed using a third analysis. The model analysis yielded, in increasing gene (FJ356265). This 18S rRNA gene, from Grant model confidence: Tamura 3-parameter model and et al. (2009), was compared among 3 Chrysophyta Gamma distributed with Invariant sites (G+I), Figure and 3 Xanthophyta. The aligned sequences were 1; Tamura-Nei model and Gamma distributed (G), 1789 bases. A model analysis for these sequences Figure 2; General Time Reversible model and suggested a Tamura 3-parameter model and Gamma Gamma distributed with Invariant sites (G+I), Figure distributed with Invariant sites (G+I). This yielded 3; and Tamura-Nei model and Gamma distributed the tree seen in Figure 6. Table 1. The characters that were examined among Ochrophyta. The states of those characters and the phyla from Appendix A are also listed. The character evolution is shown in Figure 5. Character Trait Character States Phylum Containing Character Examined Fucoxanthin in the Fucoxanthin or no Bacillariophyta, Phaeophyta, Raphidiophyta, Silicoflagellata, Eustigmatophyta, chloroplasts Fucoxanthin Pinguiophyta, Chrysophyta Symmetry Bilateral or Radial Bilateral: Bacillariophyta Symmetry Radial: Xanthophyta, Phaeophyta, Raphidiophyta, Silicoflagellata, Eustigmatophyta, Pinguiophyta, Chrysophyta Number of Flagella 1 or 2 1: Pinguiophyta 2: Xanthophyta, Phaeophyta, Raphidiophyta, Eustigmatophyta, Chrysophyta, Silicoflagellata, Bacillariophyta Results Figure 6 represents our analysis of the The overall topology of Figures 1-4 had problematic species found during our analysis of the xanthophytes as a basal group, in ochrophytes, to the taxa in question. Two major clades were observed: fucoxanthin clade. This fucoxanthin clade could be xanthophytes and chrysophytes. Phaeobotrys further divided into the basal diatoms and the solitaria was seen in the xanthophytes. polytomy of chrysophytes, pinguiophytes, eustigmatophytes, silicoflagellates, raphidiophytes, Discussion and phaeophytes. In Figures 1-4, Xanthophyta were found Figure 1 displays Bacillariophyta as a basal basal to all Ochrophyta. This was not found in group to Phaeophyta, Raphidiophyta, Silicoflagellata, analyses completed by Riisberg et al. (2009), where Eustigmatophyta, Pinguiophyta, and Chrysophyta, Bacillariophyta were determined to be basal to all with a moderate confidence bootstrap value of 67, groups analyzed and Xanthophyta were found to be with Xanthophyta appearing basal to all ochrophytes. derived. Riisberg et al. (2009) determined that This trend is seen similarly in Figures 2-4, with Xanthophyta were more closely related to bootstrap values remaining fairly consistent. Phaeophyta, as a sister taxa, and Raphidiophyta were Figure 5 represents our best estimate, using basal to this group. Riisberg et al. (2009) determined the 28S rRNA genes, of the evolution of the taxa in that Xanthophyta is more recently derived and question. All of the prior figures were similar in their closely related to Phaeophyta, as a sister taxon and topology of the taxa, however, due to low bootstrap Eustigmatophyta was basal to this group. Our values, only Bacillariophyta and Xanthophyta could analysis of the two 28S rRNA genes determined that, be placed appropriately. Bacillariophyta was basal to instead, xanthophytes were more basal and more all ochrophytes except Xanthophyta, which was basal closely related to the diatoms than to phaeophytes among these taxa. and raphidiophytes. 44 Euglena: 2013 Figure 1. The Maximum Likelihood (ML) tree was assembled here using MEGA 5.1 (Tamura et al. 2011). The model analysis here is the Tamura 3-parameter model and Gamma distributed with Invariant sites (G+I). Maximum Likelihood involves the program choosing the best tree that provides the maximum chance of producing the matrix created using the gene sequences from NCBI (see Appendix A). It is important to note the basal position of Xanthophyta to all taxa analyzed in this study. Also important is the basal nature of Bacillariophyta. It appears that there are 5 major clades to take note of in this figure. However, some of the bootstrap values (<50) say that some of the relationships may be a random placement due to uncertainty in the gene. This figure and Figures 2-4 were